Romoter and the intragenic CpG island were uncovered. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Gene. Author manuscript; available in PMC 2017 January 10. Kumar et al. Page 4 2. Materials and Methods 2.1. DNA methylation analysis using bisulfite sequencing Epididymal sperm and kidneys from adult male C57BL/6 mice were collected under protocols approved by the Miami University Institutional Animal Care and Use Committee, rinsed in phosphate ONO4059 chemical information buffered saline and either pelleted by centrifugation or homogenized using a PowerGen700 homogenizer. Genomic DNA from the sperm pellets and BHI1 biological activity kidney lysates was extracted using the 5Prime Archive pure DNA cell/tissue extraction kit according to the manufacturer’s instructions. 200 ng of genomic DNA from kidney and sperm was bisulfite treated using the EZ DNA methylation kit. Methyl Primer Express Software v1.0 was used to design primers for amplification of bisulfite treated DNA. The entire M4-CGI was amplified, in four overlapping fragments, using the primers listed in the Author Manuscript Author Manuscript Author Manuscript Author Manuscript Gene. Author manuscript; available in PMC 2017 January 10. Kumar et al. Page 5 2.3. 5-azaC and TSA treatment of cells and quantitative real time PCR Author Manuscript Author Manuscript Author Manuscript Author Manuscript GC-1spg cells were plated at 1106 cells/ml in 60 mm tissue culture dishes. The following day, the cells were treated with 5 M 5-azaC alone, 50 nM TSA alone or 5 M 5-azaC and 50 nM TSA and or their respective vehicles. Each day, fresh inhibitor or vehicle was added to the cultures and after 3 days, the cells were washed with PBS and cell pellets were harvested. DNA was extracted using 5Prime Archive pure DNA cell/tissue extraction kit. Genomic DNA from vehicle and 5 M 5-azaC treated GC-1spg cells were subjected to bisulfite sequencing as above. RNA was extracted using the TRI REAGENT method according to the manufacturer’s instructions. Following extraction, first strand cDNA synthesis was performed using the ImProm-IITM Reverse Transcription System for Two-Step RT-PCR. The cDNA was then subjected to quantitative real time PCR using the RT2 SYBR Green Fluor qPCR Mastermix on the iCycler Thermal Cycler. GAPDH was used as an internal control. Kumar et al. Page 6 Basic- M4-Promoter with BglII and HindIII and inserted into the pGL3-M4-CGI-SV40Promoter vector replacing the SV40 promoter. All DNA inserts were sequence verified before transfection. 2.5. In vitro methylation of reporter constructs Individual reporter constructs were treated with 10 units of M.SssI methylase overnight in the presence of 160 M S adenosylmethionine. Mock methylation was performed in the absence of the M.SssI methylase. The M.SssI methylase enzyme was heat inactivated at 65 C for 20 min and the DNA was ethanol precipitated before transfection into cells and assayed 
for luciferase activity. The methylation status of the constructs was verified by digestion with the methyl-sensitive restriction enzyme HpaII. 2.6. Transient transfections and dual luciferase reporter assays For dual luciferase reporter assays, GC-1spg cells, NIH3T3 and HEK 293 were plated at 2105 cells/ml in a 24 well dish and then transfected with 1 g of DNA using PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 Lipofectamine 2000. 100 ng of Renilla luciferase reporter vector pRL-TK was cotransfected to monitor transfection efficiencies. 48 h post transfection, the cells were assayed for both firefly and Renilla luciferase activit.Romoter and the intragenic CpG island were uncovered. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Gene. Author manuscript; available in PMC 2017 January 10. Kumar et al. Page 4 2. Materials and Methods 2.1. DNA methylation analysis using bisulfite sequencing Epididymal sperm and kidneys from adult male C57BL/6 mice were collected under protocols approved by the Miami University Institutional Animal Care and Use Committee, rinsed in phosphate buffered saline and either pelleted by centrifugation or homogenized using a PowerGen700 homogenizer. Genomic DNA from the sperm pellets and kidney lysates was extracted using the 5Prime Archive pure DNA cell/tissue extraction kit according to the manufacturer’s instructions. 200 ng of genomic DNA from kidney and sperm was bisulfite treated using the EZ DNA methylation kit. Methyl Primer Express Software v1.0 was used to design primers for amplification of bisulfite treated DNA. The entire M4-CGI was amplified, in four overlapping fragments, using the primers listed in the Author Manuscript Author Manuscript Author Manuscript Author Manuscript Gene. Author manuscript; available in PMC 2017 January 10. Kumar et al. Page 5 2.3. 5-azaC and TSA treatment of cells and quantitative real time PCR Author Manuscript Author Manuscript Author 
Manuscript Author Manuscript GC-1spg cells were plated at 1106 cells/ml in 60 mm tissue culture dishes. The following day, the cells were treated with 5 M 5-azaC alone, 50 nM TSA alone or 5 M 5-azaC and 50 nM TSA and or their respective vehicles. Each day, fresh inhibitor or vehicle was added to the cultures and after 3 days, the cells were washed with PBS and cell pellets were harvested. DNA was extracted using 5Prime Archive pure DNA cell/tissue extraction kit. Genomic DNA from vehicle and 5 M 5-azaC treated GC-1spg cells were subjected to bisulfite sequencing as above. RNA was extracted using the TRI REAGENT method according to the manufacturer’s instructions. Following extraction, first strand cDNA synthesis was performed using the ImProm-IITM Reverse Transcription System for Two-Step RT-PCR. The cDNA was then subjected to quantitative real time PCR using the RT2 SYBR Green Fluor qPCR Mastermix on the iCycler Thermal Cycler. GAPDH was used as an internal control. Kumar et al. Page 6 Basic- M4-Promoter with BglII and HindIII and inserted into the pGL3-M4-CGI-SV40Promoter vector replacing the SV40 promoter. All DNA inserts were sequence verified before transfection. 2.5. In vitro methylation of reporter constructs Individual reporter constructs were treated with 10 units of M.SssI methylase overnight in the presence of 160 M S adenosylmethionine. Mock methylation was performed in the absence of the M.SssI methylase. The M.SssI methylase enzyme was heat inactivated at 65 C for 20 min and the DNA was ethanol precipitated before transfection into cells and assayed for luciferase activity. The methylation status of the constructs was verified by digestion with the methyl-sensitive restriction enzyme HpaII. 2.6. Transient transfections and dual luciferase reporter assays For dual luciferase reporter assays, GC-1spg cells, NIH3T3 and HEK 293 were plated at 2105 cells/ml in a 24 well dish and then transfected with 1 g of DNA using PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 Lipofectamine 2000. 100 ng of Renilla luciferase reporter vector pRL-TK was cotransfected to monitor transfection efficiencies. 48 h post transfection, the cells were assayed for both firefly and Renilla luciferase activit.
